Isolation and Characterization of Thermostable Alpha-Galactosidases for Application of High-Temperature Processing of Soy Molasses

The α-Galactosidases from the novel thermophilic strain KM-THCJ and hyperthermophilic Thermotoga neapolitana were examined for their potential use in processing of soy oligosaccharide waste by-products. The α-galactosidase of strain KM-THCJ has been purified to homogeneity and has a MW of ca. 176 kDa and a subunit MW of ca. 88 kDa occurring as a dimer, as determined by gel filtration (SDS-PAGE ca. 80 kDa). The enzyme exhibits a pH optimum of 8.0 and demonstrates broad pH stability. The optimum temperature of enzyme activity was 77.5$\sp\circ$C, with a demonstrated temperature stability up to 70$\sp\circ$C. Known sulfhydryl groups inhibitors decrease enzyme activity. Attempts have been made to clone the Strain KM-THCJ α-galactosidase, providing numerous positive results, however, no expressing clone has been obtained. A cosmid library of T. neapolitana has been screened for α-galactosidase expression. One cosmid clone, RU72, was shown to express high levels of α-galactosidase activities. Examination of crude extracts of RU72 demonstrated an α-galactosidase temperature optimum ranging from 93 to 97$\sp\circ$C and temperature stability up to 84$\sp\circ$C. Restriction enzyme digested cosmid insert was sub-cloned and sequenced, elucidating an α-galactosidase gene. The α-galactosidase gene demonstrated nucleic and amino acid sequence similarities to the α-galactosidases of Thermoanaerobacter ethanolicus, Pediococcus pentosaceus, Streptococcus mutans, and Escherichia coli. The α-galactosidase appears to exist in an operon containing galactose transport and transfer genes. The putative α-galactosidase gene was PCR amplified and inserted into pCR2.1 which expressed a thermostable α-galactosidase. A 61 kDa protein was observed in partially purified extracts, similar to the deduced size of the putative gene. Both bacterial strains produce α-galactosidases which hydrolyze soy α-galactosides. The α-galactosidase from Thermotoga neapolitana, stable at 80$\sp\circ$C, meets the criteria required for use in soy processing. The moderately stable α-galactosidase from Strain KM-THCJ may have applications for use in the sucrose industry due to increased activity and stability at moderate alkaline pH.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science and Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• King, Michael Roy

Contributors dc:contributor

• Bruce M. Chassy

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(MiAaPQ)AAI9812655

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/83723